Mice with genetically altered expression of costimulatory molecules CD80 (B7-1), CD86 (B7-2), and CD40, or of the costimulatory receptors CD28 and CD154 (CD40 ligand) have been analyzed for thymic development and T cell repertoire. Our findings identify a previously unappreciated role of redundant costimulatory pathways mediating an essential function in thymic development. The effect of these costimulatory pathways on selection of the T cell repertoire was studied in parallel. Two pathways of negative selection have been identified. One pathway is CD40L-dependent and acts at a relatively early stage in intra-thymic development and is mediated by a non-cell-autonomous mechanism. The second is CD40L-independent and occurs later in intra-thymic or post-thymic development. Interestingly, we have observed that when negative selection is abrogated by inactivation of the CD40L pathway, functional self tolerance is maintained by a non-deletional mechanism. This non-deletional tolerance is mediated by a CD28-dependent pathway. Thus, redundant pathways exist to protect against self reactivity in the thymus, and disruption of both C40L (deletional) and CD28 (non-deletional) mechanisms of self tolerance results in a population of highly self-reactive thymic T cells. We have analyzed the signals that mediate T cell-epithelial cell cross-talk during thymic development. We have observed that CD28 costimulation is essential for optimal induction of TNFab in single-positive (SP) thymocytes, and that combined defects in CD40-CD40L and TNFab pathways result in defects in medullary thymic eipthelial cell (mTEC) development that are similar to those in combined CD40-CD40L and CD28-B7 disruption, and are in fact as profound as those seen in complete absence of mature SP TCRab thymocytes. These findings indicate a novel role for CD28-B7 in addition to other mediators of T cell-TEC cross-talk and demonstrate that any and all signals provided by mature thymocytes for mTEC development are provided by activation of RelB-dependent alternative NFkB signaling. Generation of the T cell repertoire requires positive selection of cells expressing TCR capable of recognizing foreign antigens, but negatively selected to eliminate cells with potentially dangerous specificity against normal self antigens. The function of costimulatory CD28-B7 and CD40-CD40L pathways in this repertoire selection has not been fully elucidated. We have initiated studies using peptide-MHC tetramers to identify antigen-specific T cells, and have generated the first conditional knockouts for B7 and CD40 to analyze cell type-specific function of these costimulatory pathways in repertoire selection. Generation of the T cell repertoire requires positive selection of cells expressing TCR capable of recognizing foreign antigens, but negatively selected to eliminate cells with potentially dangerous specificity against normal self antigens. The function of costimulatory CD28-B7 and CD40-CD40L pathways in this repertoire selection has not been fully elucidated. We have used peptide-MHC tetramers to identify antigen-specific T cells, and have generated the first conditional knockouts for B7 and CD40 to analyze cell type-specific function of these costimulatory pathways in repertoire selection.Thymic central tolerance is critical for the prevention of autoimmunity. However, the underlying molecular and cellular mechanisms mediating this tolerance are not fully understood. We have analyzed the requirements for B7-CD28 co-stimulation and for B7 expression by specific antigen-presenting cell (APC) types for thymic clonal deletion and Treg cell generation of endogenous tissue-restricted-antigen (TRA)-specific thymocytes. We have employed p-MHC tetramers to identify and track T cells with specificity for specific endogenous tissue-restricted antigens and our recently generated conditional B7 knockout mouse strains to identify APC requirements for Treg generation and for clonal deletion of TRA-reactive cells. Our findings determined that: 1) both clonal deletion and Treg cell generation of TRA-specific thymocytes are B7-CD28-dependent; 2) clonal deletion and Treg cell generation differ in their CD28 signaling domain requirements ; and 3) the role of B7-expressing dendritic cells (DC), B cells, and thymic epithelial cells (TEC) can differ in B7-dependent clonal deletion versus Treg cell generation. In the absence of B7-CD28 co-stimulation, mature TRA-specific Tconv cells populated the periphery in increased numbers and were capable of mediating destructive autoimmunity. Our findings reveal a previously unappreciated role of B7-CD28 co-stimulation in shaping the T cell repertoire through thymic clonal deletion and Treg cell generation with distinct requirements of CD28 signaling and B7-expressing APC. The ability to generate a rapid and sustained T cell response to external pathogens and transformed cancer cells is essential for protection of the host. At the same time, deletion of autoreactive T cells during development and repression of excessive or autoreactive responses in peripheral tissues is essential to proper T cell protective function. This duality in regulation of T cell function is accomplished by two types of T cells: conventional T cells (Tconv) that provide helper (CD4+) and killer (CD8+) functions, and regulatory T cells (Treg) that control Tconv T cell-dependent responses. Treg cells have been assigned to two subsets based on the origin of their generation: thymic (or natural) Treg (tTreg) that develop in the thymus and peripheral Treg (pTreg) generated in periphery from Tconv cells under specific conditions. The development of tTreg appears to require both TCR and other factors such as costimulatory receptors and cytokines, but the precise mechanisms of tTreg generation have not been fully elucidated. We addressed the role of TCR sequence in determining whether T cells develop into tTreg or Tconv lineages. We carried out a comprehensive comparison of both TCRa and TCRb sequences of thymic tTreg and Tconv cells using a UMI based 5' smart switch method. This comparison revealed that, although many sequences were unique to either Treg or Tconv, a substantial proportion of TCaR (14-20%) and TCRb (8-22%) sequences from tTreg were also found in Tconv cells of two normal mouse strains. TCRa analysis of a TCRb transgenic mouse line revealed an even higher proportion (71%) of sequences found in tTreg that were also found in Tconv cells. Interestingly, these shared TCRa clonotypes that were common to tTreg and Tconv cells were significantly more abundant than non-shared TCRa sequences of tTreg and Tconv cells. Finally, we used machine learning to develop an algorithm that was capable of distinguishing non-shared TCRa and TCRb sequences expressed by tTreg from those of Tconv cells and in addition found that specific amino acid trimers were differentially expressed in either tTreg or Tconv cells. Taken together, our findings identify TCR sequence characteristics that bias to tTreg or Tconv fate, in addition to factors that can drive cells with identical TCR sequence into either Tconv or tTreg lineages. These findings identify two populations of tTreg, one in which Treg fate is determined by unique properties of the TCR, and another with TCR properties characteristic of Tconv cells, with tTreg determined by TCR-independent factors.